Recent findings have shown that the extraordinary level of polyamine biosynthesis found in prostate cells, and accentuated in prostate cancer (CaP), places strain on one-carbon metabolism and the methionine cycle making prostatic epithelial cells highly sensitive to perturbation of these metabolic pathways. The high level of polyamine biosynthesis is driven by the activity of spermidine/spermine N1-acetyltransferase (SSAT), which acetylates the polyamines leading to their secretion into the lumen, and necessitates de novo synthesis of polyamines to maintain intracellular levels. The methionine salvage pathway (MSP) provides a means of mitigating this metabolic strain by recycling the one-carbon unit lost to polyamine biosynthesis back into the methionine cycle, thereby replenishing s-adenosylmethionine (SAM) pools and protecting nucleotide pools. While this pathway is compromised in many types of cancer by deletion of the methylthioadenosine phosphor- ylase (MTAP) gene (located within 100kb of the p16 locus), deletion of MTAP is very rare in CaP. Our central idea is to increase polyamine biosynthetic flux and the associated stress by upregulating SSAT activity, while at the same time interfering with the cells' ability to mitigate the stress by inhibiting the MSP. The objective of the current proposal is to determine the therapeutic potential of polyamine catabolism upregulation MSP inhibition, either alone or in combination, to enhance the extent and/or duration of clinical benefit of androgen deprivation therapy (ADT). The central hypothesis is that the MSP is critical to CaP due to high metabolic flux through polyamine biosynthesis, and that this dependence can be enhanced by increasing the activity of SSAT. Targeting these pathways will provide novel therapeutic strategies to prevent or delay recurrence during ADT. Therefore, we propose to use a combination pharmacological approach to increase metabolic stress of CaP cells by treating with a polyamine analogue to upregulate polyamine catabolism by increasing SSAT activity, while at the same time reducing the cells' ability to mitigate that stress by inhibiting MTAP. The hypothesis will be tested by pursuing three specific aims: In Aim 1 we seek to determine the potential for synergistic or additive relationships between MSP inhibition and enhancement of polyamine catabolism to create a metabolic crisis and to understand the mechanistic basis of drug effects. In Aim 2 we will evaluate the efficacy of generating a metabolic crisis to treat established androgen independent CaP in human cell line xenografts. In Aim 3 we will determine the effectiveness of generating a metabolic crisis to prevent castration recurrence models of progression to ADT-recurrent CaP. This novel approach takes advantage of an inherent metabolic strain accentuated in prostatic epithelial cells in order to develop novel therapeutic strategies. These pre-clinical studies will determine if therapeutic efficacy can be obtained through leveraging of already existing metabolic strain by adding to it (increasing polyamine catabolism) while simultaneously blocking a critical salvage pathway that helps to mitigate that strain.